The skin is a tissue made up of two parts, the epithelium or external part and the dermis, or the internal part on which the epithelium is positioned. These two parts have clearly different characteristics. There is practically no extracellular tissue in the epidermis, whereas this component is clearly predominant over cells in the dermis. The skin is a tissue that can be re-constructed by tissue engineering techniques (Parenteau N, Sci Am, 280: 83-84, 1999). In these techniques, in general, the cellular component is generated “ex vivo” by cell cultivation techniques. These techniques, starting with a small number of cells taken from a small skin biopsy, obtain a large number of cells in a short time. These “ex vivo” expanded cells can be used to build large areas of artificial skin. The extracellular matrix can not be produced by cell cultivation, but it is previously designed and manufactured outside the body. The extracellular matrix has to be capable of providing structures that facilitate the adhesion of the previously cultivated dermal cells and stimulating the normal growth of these cells. On this artificial matrix, the cells begin to manufacture the normal proteins that make up the natural dermal matrix, and at the same time, they slowly degrade the original structure, so that over time this artificial matrix is replaced by a true extracellular matrix completely similar to a natural one. The previously cultivated epithelial cells (Keratinocytes in the case of skin) can be seeded on this artificial matrix, where with new cell cultivation techniques, these cells are capable of generating a structure that is very similar to the normal epithelium from which they originated. In other words, one of the key factors in skin tissue engineering is the design of dermal matrices that imitate the body's natural conditions as much as possible, and where the cells introduced are capable of starting a complex process, the purpose of which is to develop a structure as similar as possible to natural skin. The other key factor in skin tissue engineering is the capacity of the dermal matrix to facilitate the growth of the cells that are seeded on it. The development of dermal matrices that encourage the growth of both dermal and epidermal cells would mean that it would be possible to cultivate large areas of artificial skin from a minor biopsy. This is especially important when the artificial skin is for treating major burns, where up to 90-95% of the body's total surface area has to be replaced as quickly as possible, using the small areas of health skin that remain on the patient. The lack of dermal matrices capable of generating these large areas of artificial skin from minor biopsies is one of the limitations of previously described dermal matrices (Sheridan R and Tompkins, Burns 25: 97-103, 1999).
There are several artificial dermis models. Some of the previously used models are described briefly below:
Collagen type I of animal origin, since this is the most abundant of the proteins present in the dermis (Maraguchi T et al. Plast Reconstr Surg, 93: 537-544, 1994, Muhart et al. Arch Dermatol, 135: 913-918, 1999).
Chondroitin sulphate (Boyce S et al. Surgery, 103: 421-431, 1988).
Nylon associated or not to an impermeable Sylastic membrane (Naughton and Mansbridge, Clin Plast Surg, 26: 579-586, 1999).
Poly-lactide/poly-glycolide (Giardino et al, J Trauma 47: 303-308, 1999). These polymers form a web that acts as a structure where the fibroblasts take, grow and are capable of segregating normal dermal matrix proteins.
Fibrin. This protein, the precursor of which, fibrinogen, is obtained from human plasma, has been used in different ways in the cultivation of keratinocytes. Fibrin provides a good base for the growth of epithelial cells, so this protein has been used as an inert support on which to grow cheratinocytes (Ronfard et al, Burns 17: 181-184, 1991) (Broly et al, ES2060803). Fibrin does not interfere with the later development of a correct dermal/epidermal binding between the bed of the injury and the cultivated keratinocytes. Because of these characteristics, fibrin has been broadly used as a transport system for keratinocytes (Pellegrino et al. Transplantation 68: 868-879, 1999, Kaiser and Stark, Burns 20: 23-29, 1994).
Fibrin and/or the gels made after the clotting of human plasma proteins have been used as a vehicle for transplanting skin cells previously expanded “in vitro” (Sadaki I, JP10277143).
Fibrin can also be used as a dermal base for the production of large areas of cultivated skin (Meana et al, Burns 24: 621-630, 1998). The fibroblasts embedded in fibrin gels are capable of growing. At the same time, these fibroblasts act like authentic inducers of keratinocyte growth, so that platting a very limited number of cultivated keratinocytes on a gel made up of fibrin and fibroblasts, in 8-12 days we obtain a stratified confluent epithelium that imitates the normal human epithelium. This capacity of fibrin gels for the development of epithelial cells has been used in another artificial skin model (Meana et al, P9701533). Moreover, fibrin can be used in the presence of other components that increase its rigidity and facilitate its use as a dermal support (Meana A. P9601684). This capacity of fibrin gels and fibroblasts to obtain large areas of artificial skin from a minor skin biopsy is lacking in models based on artificial dermis with other compositions. The explanation of this lies in the fact that fibrin-based gels are able to imitate the physiological wound-repairing mechanism (Martin P, Science 276: 75-81, 1997).
However, the production of the dermal matrix based on fibrin concentrates is only an imitation of the physiological process. The true fibrin clot that is formed as part of the tissue repair and defence mechanism is at the expense of blood plasma. There are many proteins in the extracellular blood fraction, and one of them, fibrinogen, is the soluble precursor of fibrin, the main but not the only protein in the clot. The leak of the plasma after the aggression of a tissue is one of the ways in which the entire clotting process is started. When the aggression occurs and the tissue products come into contact with the skin, the so-called extrinsic clotting pathway is activated, and the final result if the activation of the inactive thrombin precursor present in plasma. This thrombin starts converting the fibrinogen into fibrin and eventually into soluble fibrin which, bound to blood cells, forms part of the fibrin clot, the first step in curing and later repairing a lesion to the body (Singer and Clark, N Engl J med. 341: 736-746, 1999). Of the cells involved in forming the clot, special mention should be made of the platelets. These cells are an important deposit for cytokines, the substances responsible for initiating the cell response in the final repair process for wounds. Platelets are also involved in the development of the fibrin clot inside the veins. This is called the intrinsic clotting pathway, in which a stimulus provokes the development of platelet aggregation that will activate a series of plasmatic proteins that, in turn, will stimulate others by a cascade process mechanism. Finally, we will have the thrombin that will start forming the clot. In both processes, extrinsic and intrinsic clotting pathways, the presence of free calcium ions is essential to complete their development, because some of these pathways' proteins depend on this ion to be activated. After the fibrin clot is formed from the blood plasma, the cytokines, initially released by the platelets, will attract other cells, such as macrophages, neutrophyls, etc., which will start to destroy the clot and replace this fibrinoid tissue by the normal tissue that existed prior to the aggression. These cells, in turn, will manufacture other cytokines that will maintain and control the response to the aggression. They will attract dermal fibroblasts and endothelial cells to the wound, which will complete the repair response. These new repairing cells will manufacture other cytokines that will attract epithelial cells to the wound to cover its entire surface. In turn, epithelial cells are capable of manufacturing many substances that provoke different cell responses in the underlying dermal cells. Epithelial cells also have a catabolic effect on the fibrin clot, because they need to penetrate and eliminate it in order to re-coat the surface of the wound (Singer and Clark, N Eng J. Med. 341: 738-746, 1999). Once the lesion has been covered by the epithelium, the curing process is complete.
We can consider that the physiological repair of a wound is based on a fibrin clot rich in plasmatic cytokines, based on the fibrinogen dissolved in the plasma. This clot will start the body's primary repair response and will induce the nearby epithelial cells to migrate from the points closest to the wound and finally close it. This repair process has limits, and when the injury completely destroys all the epithelial cells present (broad and deep burns), an amount of artificial epithelium is required, either by means of grafts of cultivated keratinocytes, suspended keratinocytes or cultivated artificial skin, for the process to be completed (Naysaria et al. TIBTECH 13: 91-100, 1995).
If the origin of wound repair is in the fibrin clot from plasma, it is possible for an artificial skin model based on the use of human plasma as the primary source of the extracellular matrix to be highly effective and provide extraordinary encouragement for cell growth, since it reconstructs the physiological conditions of the body's wound repair process.